KR20200129596A - Composition for Preventing or Treating of Degenerative Brain Disease Comprising Extract of Zanthoxylum piperitum Fruit - Google Patents

Abstract

The present invention relates to a composition for preventing and treating degenerative brain diseases which contains a Zanthoxylum piperitum fruit extract as an active component. More specifically, the present invention relates to a composition for preventing and treating degenerative brain diseases containing a Zanthoxylum piperitum fruit extract, extracted by using ethanol as a solvent, as an active component. The Zanthoxylum piperitum fruit extract of the present invention can regulate nerve inflammation by inhibiting the production of inflammatory factors including NO, iNOS, and COX, inhibiting the production of inflammatory cytokines including IL-1β, IL-6, and TNF-α, and inhibiting the activity of NF-κB, thereby being able to be usefully used for preventing and treating degenerative brain diseases.

Description

Composition for Preventing or Treating of Degenerative Brain Disease Comprising Extract of Zanthoxylum piperitum Fruit}

The present invention relates to a composition for the prevention and treatment of degenerative brain diseases containing an extract of Chopi tree fruit as an active ingredient, and more particularly, prevention of degenerative brain diseases containing an extract of Chopi tree fruit extracted using ethanol as a solvent as an active ingredient And to a composition for treatment.

Degenerative brain disease refers to a disease that occurs in the brain among degenerative diseases that occur with age, and can be classified by considering the main symptoms and the invading brain region, representatively including Alzheimer's disease and Parkinson's disease. . Degenerative brain disease is known to be caused by neurodegeneration due to aging and neuronal cell death due to protein aggregation due to genetic and environmental factors, but the exact cause is not yet known, so basic research to determine the cause is actively conducted. It is a situation, and it is known that neuroinflammation always increases when a person has degenerative brain disease.

In Korea, which is rapidly aging, the development of treatments for degenerative brain diseases is urgent. By 2026, it is predicted to reach an ultra-aged society over 20%, and the problem of degenerative brain disease is expected to rise seriously not only in Korea but also around the world. It is estimated that the number of dementia patients in Korea will be from 470,000 in 2010 to 750,000 in 2020, and cerebrovascular disease has remained the second largest cause of death in Korea for the past 10 years.

Parkinson's Disease (PD) is a chronic disease caused by a lack of a neurotransmitter called dopamine in the brain, as the main symptoms include tremor, stiffness, slow behavior, and postural dysfunction. Dopamine is produced by neurons called Substantia nigra pars compacta (SNpc) in the brain, and neurons in black matter are intricately connected to the motor cortex and other parts of the brain so that the body's movements can be performed smoothly, harmoniously and accurately. Haeju is connected to a site called the basal ganglia. Parkinson's disease is caused by a lack of dopamine, a substance secreted to control the function of the basal ganglia in the black matter.

Symptoms of Parkinson's disease can be largely divided into primary and secondary symptoms. The primary symptoms are stiffness, tremors, slow or decreased body movements, and imbalances in the body and impaired walking. Secondary symptoms refer to symptoms derived from primary symptoms or caused by invasion of the nervous system other than black matter.

Currently used treatments for Parkinson's disease include drug therapy, surgical therapy, and physical therapy. In the case of drug therapy, it supplements dopamine, which is generally insufficient in the brain, and neurotransmitter imbalance due to lack of dopamine. Drugs for the purpose of preventing or delaying the destruction of nerve cells and controlling other symptoms such as depression are used, for example, amantadine, anticholinergic drugs, L-dopa, Cinemet, Madopa, dopamine Agonists, eldefril and antidepressants. However, since these drugs cannot revive dead neurons, they have limitations in that they are not intended to cure but control symptoms.

Alzheimer's is known as the most common degenerative brain disease. It is usually chronically progressive, and shows progressive decline in intellectual functions such as memory, judgment, and language skills, and impairments in daily life ability, personality, and behavior. Alzheimer's accounts for about half of the causes of dementia. As the most common dementia, it accounts for about half of all dementia. This dementia is a disease in which a neurotransmitter called acetylcolline is reduced due to damage to normal cells, resulting in loss of memory, language function, and judgment, and a change in personality, eventually losing the ability to take care of itself.

Regarding the treatment of Alzheimer's disease, acetylcholinesterase inhibitors are recognized for their effectiveness in mild and moderate Alzheimer's disease, but ingredients that are officially recognized by the US Food and Drug Administration and widely used include tacrine and donepezil. , Rivastigmine, galantamine, etc. are known to show liver toxicity and are rarely used.

Therefore, there is a need to develop a therapeutic agent for degenerative brain cells derived from natural substances that do not exhibit side effects. Considering the characteristics of neurons in the brain, there is no side effect, so it can effectively regenerate nerve cells among natural substances that can be continuously consumed. The necessity of developing substances that can increase and preventing degenerative brain diseases using them is newly recognized.

As extracts for treating nervous system diseases, there are pomegranate, zinnia, green tea, blueberry, etc., and a patent has been disclosed that can treat Alzheimer's disease, Huntington's disease or stroke using Nerium or Thevetia ( Republic of Korea Registration No. 10-1046126; International Publication No. WO2012-071152).

On the other hand, Zanthoxylum piperitum is a deciduous shrub of the Unhyang family, and is usually used as a spice and medicine for the fruit bark, and seeds, young leaves, trees and stems are also used for various purposes. Chopi fruit is used as a detoxification, anthelmintic, and pain reliever in herbal medicine, and is mainly distributed in Korea, Japan and China. In the prior invention, it has been reported that mainly chopidae extract exhibits effective antiviral, insecticidal, anticancer, osteoporosis, and skin whitening activity (Korean Patent No. 10-0314545; Korean Patent No. 10-0666299; Korean Patent No. 10) -0883992; Korean Registered Patent No. 10-1182824).

Accordingly, the present inventors have endeavored to develop a composition for the prevention and treatment of degenerative brain diseases, as a result of which the Chopi tree fruit extract inhibits the expression of nitrogen monoxide (NO) and inflammatory mediators without cytotoxicity, as well as NF-κB ( It was confirmed that the nuclear factor kappa-light-chain-enhancer of activated B cells) effectively inhibited the activity, and the present invention was completed.

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating degenerative brain diseases containing an extract of Chopi tree fruit as an active ingredient.

Another object of the present invention is to provide a health functional food composition for preventing and improving degenerative brain diseases containing the chopidae fruit extract as an active ingredient.

To achieve the above object,

The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing chopi tree fruit extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing and improving degenerative brain diseases containing the Chopi tree fruit extract as an active ingredient.

In a preferred embodiment of the present invention, the chopidae fruit extract may be extracted using water, a lower alcohol having 1 to 4 carbon atoms, or a mixture thereof as a solvent.

In another preferred embodiment of the present invention, the chopidae fruit extract may be extracted using ethanol as a solvent.

In another preferred embodiment of the present invention, the degenerative brain disease is dementia, Alzheimer's disease, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis. ) And Parkinson disease (parkinson disease) may be any one selected from the group consisting of.

In another preferred embodiment of the present invention, the chopidae fruit extract may inhibit the production of inflammatory factors including NO, iNOS and COX-2.

In another preferred embodiment of the present invention, the chopi tree fruit extract may inhibit production of inflammatory cytokines including IL-1β, IL-6 and TNF-α.

In another preferred embodiment of the present invention, the chopi tree fruit extract may inhibit the activity of NF-κB through inhibition of IκB-α phosphorylation.

Chopi tree fruit extract of the present invention inhibits the production of inflammatory factors including NO, iNOS and COX-2, inhibits the production of inflammatory cytokines including IL-1β, IL-6 and TNF-α, and inhibits the activity of NF-κB. Since nerve inflammation can be controlled through, it can be usefully used for preventing and treating degenerative brain diseases.

1 is a schematic diagram for the prevention and treatment of degenerative brain diseases through the control of neuroinflammation of Chopi tree extract.
2 is data showing the NO generation inhibitory efficacy and cytotoxicity of the Chopi tree fruit extract according to the extraction solvent.
3 is a data showing the NO generation inhibitory effect and the degree of cytotoxicity when treated with ethanol extract of Chopi tree fruit on BV-2 microglia stimulated with LPS.
4 is data confirming the expression levels of iNOS and COX-2 proteins, which are inflammatory mediators, when treated with ethanol extract of Chopi tree fruit on BV-2 microglia stimulated with LPS. The lower graph is data showing the relative amount of expression for β-actin by quantifying the amount of each protein.
FIG. 5 shows the inflammatory response mediators including iNOS and COX-2 and IL-1β, IL-6, and TNF-α when treated with ethanol extract of Chopi tree fruit on BV-2 microglia stimulated with LPS. This is the data confirming the level of mRNA expression of inflammatory cytokines.
6 is data confirming the degree of phosphorylation of IκB-α, a protein regulating NF-κB activity, when treated with ethanol extract of Chopi tree fruit on BV-2 microglia stimulated with LPS.
Figure 7 is a chopi tree ( Zanthoxylum) on BV-2 microglia stimulated with LPS piperitum ) leaf extract, Zanthoxylum coreanum ) Data showing the inhibitory effect of NO generation and the degree of cytotoxicity of leaf extracts and extracts of C. coreanum .
8 is a data showing the NO generation inhibitory effect and the degree of cytotoxicity when treated with the chopi tree leaf extract and the chopi tree stem extract on BV-2 microglia stimulated with LPS.
9 is a data showing the NO generation inhibitory effect and the degree of cytotoxicity when treated with LPS-stimulated BV-2 microglia with a leaf extract and a stem extract of a tree plant.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing chopi tree fruit extract as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing and improving degenerative brain diseases containing the Chopi tree fruit extract as an active ingredient.

The chopidae fruit extract may be extracted using water, a lower alcohol having 1 to 4 carbon atoms, or a mixture thereof as a solvent, and the lower alcohol is preferably ethanol or methanol. Most preferably, the chopi tree fruit extract may be characterized in that it is extracted using ethanol as a solvent.

In the present invention, the degenerative brain diseases are dementia, Alzheimer's disease, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, and Parkinson's disease. disease) may be characterized in that any one selected from the group consisting of.

In the present invention, the chopi tree fruit extract

I) inhibition of production of inflammatory mediators including NO, iNOS and COX-2;

Ii) inhibition of production of inflammatory cytokines including IL-1β, IL-6 and TNF-α; And

Iii) inhibition of NF-κB activity through inhibition of IκB-α phosphorylation; it may be characterized by regulating nerve inflammation.

1 is a schematic diagram for the prevention and treatment of degenerative brain diseases through the control of neuroinflammation of the Chopi tree extract, in the present invention, whether it is possible to prevent or treat degenerative brain diseases through the neuroinflammation control and neuroprotective action of the Chopi tree fruit extract Confirmed.

In a specific embodiment of the present invention, the dried product of chopi berry was extracted using hot water (80°C) and ethanol, respectively, and 65.58 g and 73.38 g of dried product were obtained from 1 kg of sample through a freeze-drying process.

In addition, as shown in Figure 2, as a result of treating the hot water extract of BV-2 microglia stimulated with LPS and the ethanol extract of Chopi fruit by concentration, it was confirmed that both extracts suppressed NO (nitric oxide) generation The IC ₅₀ value was found to be 459.33 μg/ml for hot water extract and 189.11 μg/ml for ethanol extract.

That is, it was confirmed that the ethanol extract inhibited NO generation more effectively than the hot water extract of Chopi fruit, and it was confirmed that the ethanol extract of Chopi fruit was more suitable for the prevention and treatment of degenerative brain diseases.

In another specific embodiment of the present invention, when the ethanol extract was treated on BV-2 microglia stimulated with LPS, inhibition of NO generation and cellular activity were confirmed. As a result, as shown in FIG. 3, it was confirmed that the ethanol extract of Chopi in the 20 ~ 200 ㎍ / ㎖ interval did not affect the cell activity, it was confirmed that also effectively inhibited NO generation.

In another specific embodiment of the present invention, when treated with ethanol extract of Chopi tree fruit on BV-2 microglia stimulated with LPS, as shown in FIG. 4, iNOS and COX-2 proteins, which are mediators of inflammatory response It was confirmed that the expression decreased depending on the concentration of the ethanol extract of Chopi fruit.

As shown in Figure 5, mRNA expression of inflammatory mediators including iNOS and COX-2 and inflammatory cytokines including IL-1β, IL-6, and TNF-α is also reduced in a concentration-dependent manner by treatment with ethanol extract of Chopi fruit I confirmed that.

In addition, as shown in Figure 6, it was confirmed that the phosphorylation of IκB-α, a modulator of NF-κB, an important transcription factor of inflammatory mRNA, decreases in a concentration-dependent manner by treatment with ethanol extract of Chopi fruit.

In other words, it was confirmed that the extract of Chopi tree fruit reduces neuroinflammation by inhibiting the production of inflammatory factors including NO, iNOS and COX-2, and the production of inflammatory cytokines including IL-1β, IL-6 and TNF-α. In addition, it was confirmed that neuroinflammation was controlled by inhibiting the mRNA expression of inflammatory factors by finally inhibiting the activity of NF-κB through inhibition of IκB-α phosphorylation.

Furthermore, in another specific embodiment of the present invention, in order to confirm the excellence of the chopi tree fruit extract of the present invention, the same chopi tree ( Zanthoxylum piperitum ), as well as other sites (leaves and stems) other than the fruit, as well as the cytotoxic and anti-inflammatory efficacy of leaf and stem extracts of Zanthoxylum coreanum . As shown in FIG. 7, it was confirmed that the Chopi tree leaf extract, the Chopi tree leaf and the stem extract suppressed NO generation, but showed toxicity to BV-2 cells. In addition, when the Chopi tree leaf extract and Chopi tree stem extract were treated by concentration (FIG. 8), in particular, it was confirmed that the Chopi tree leaf extract showed remarkable cytotoxicity, and the Chopi tree leaf extract and the Chopi tree stem extract were also at high concentration. It was confirmed that it showed cytotoxicity (FIG. 9).

On the other hand, as shown in Figs. 2 and 3, the chopi tree fruit extract of the present invention has been confirmed to exhibit effective anti-inflammatory activity without cytotoxicity at a high concentration, so the chopi tree fruit extract is a leaf or stem extract of the chopi tree and the chopi tree Compared to that, it has an effect that can be applied as a treatment for degenerative brain diseases or a treatment for neurodegenerative diseases without side effects.

Each of the pharmaceutical compositions of the present invention can be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and may be formulated in the form of external preparations, suppositories and sterile injectable solutions. It may further include a pharmaceutically acceptable carrier, excipient, and diluent according to each formulation. In addition, it may be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions according to a conventional method.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating or formulating the pharmaceutical composition, it is prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the composition, such as starch, calcium carbonate, and sucrose. , Lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used. The above ingredients may be added independently or in combination to the active ingredient, that is, the fraction of P.

As used herein, the term "administering" means providing a pharmaceutical composition of the present invention to an individual by any suitable method. The pharmaceutical composition of the present invention is an amount of an active ingredient or a pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human considered by a researcher, veterinarian, doctor or other clinician, that is, relief of symptoms of the disease or disorder to be treated. It can be administered in a therapeutically effective amount, which is an amount that induces. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, the patient's age, weight, and general health condition. , Sex and diet, administration time, administration route and composition ratio, treatment period, and various factors including drugs used simultaneously. The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, intravenous, intraperitoneal, intramuscular, intraarterial, oral, intracardiac, intramedullary, intrathecal, transdermal, intestinal, subcutaneous, sublingual, or topical administration, but is not limited thereto. The pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, and may be administered once a day or divided into several times.

Chopi fruit extract of the present invention can be used as a health functional food, food additives or dietary supplements. When the extract of the present invention is used as a food additive, the extract may be added as it is, or may be appropriately used according to a conventional method, such as used by mixing with other foods or food ingredients.

In addition, the mixing amount of the chopi tree fruit extract may be appropriately changed according to the purpose of use (prevention, health or therapeutic treatment). As a specific example, when preparing food or beverage, the extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for health control purposes, it may be added in an amount below the above range, and since there is no problem in terms of safety, the active ingredient can be used in an amount above the above range. have.

There is no particular limitation on the kind of the food, but examples of foods to which the extract of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. There are dairy products, various soups, beverages, teas, drinks, alcoholic beverages, vitamin complexes, etc., and all health foods in the usual sense are included.

When the food composition of the present invention is prepared as a beverage, it may include additional ingredients such as various flavoring agents or natural carbohydrates, like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

The food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid. It may include a carbonation agent used in beverages, and may include flesh for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage, but is not limited thereto. These components may be used independently or in combination. The ratio of the additive is not largely limited, but it is preferably contained within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Preparation of Chopi tree fruit extract and confirmation of anti-inflammatory activity

As for the barberry fruit, dried barberry fruit was purchased at a grocery store and extracted using hot water extraction method and ethanol extraction method, respectively.

First, 10 L of distilled sugar was added to 1 kg of dried chopi berry, boiled at 80° C. for 15 hours to extract the extract, and then lyophilized to obtain a total of 65.58 g of hot-water extract of Chopi fruit.

Next, 10 L of 100% ethanol (alcohol) was added to 1 kg of the dried chopi berry, and then reacted for 15 hours, and then lyophilized to obtain a total of 73.38 g of the ethanol extract of Chopi fruit.

Confirmation of anti-inflammatory efficacy of Chopi tree fruit extract according to extraction method

2-1 Cytotoxicity check

In the present invention, the degree of cytotoxicity for each of the hot water extract and ethanol extract obtained in Example 1 was measured. BV-2 cell viability was measured using a known method (Kim, JJ, et al. , Journal of Ethnopharmacology , 140:213-221, 2012).

First, the murine microglia cell line BV-2 was used in the experiment, and the BV-2 cells were heat-inactivated with 10% FBS and antibiotics (penicillin 100 U/ml, streptomycin 100 μg/). Ml) was prepared by culturing using DMEM medium containing (37° C., 5% CO ₂ and 95% air).

Then, BV-2 cells were plated in a 96-well plate at 1×10 ⁴ cells/well, and then samples having different concentrations were treated. After 1 hour, treatment with LPS (200 ng/ml), which is a neuroinflammation inducing factor, was then stimulated for 24 hours, and after treatment, the medium was discarded and 100 µl DMEM medium containing MTT (500 µg/ml) Was added to each well and incubated at 37°C for 4 hours. After incubation, the medium was discarded and DMSO was added to each well to dissolve formazan. Optical concentration (OD) was measured at 540 nm and compared with the control.

To analyze cytotoxicity, statistical analysis of the samples was performed by one-way ANOVA (post-hoc Tukey's multiple comparison test), and the results for all experiments were expressed as mean ± standard deviation.

Cytotoxicity by Concentration of Chorus Fruit Extract Hot water extract Ethanol extract Addition amount (㎍/㎖) LPS (200ng/ml) Cell viability (%) Addition amount (㎍/㎖) LPS (200ng/ml) Cell viability (%) - - 100±1.2 - - 100±1.9 - + 88.9±1.5 - + 91.0±2.0 20 + 89.5±3.9 20 + 89.1±3.8 100 + 89.6±3.2 100 + 90.0±3.4 200 + 89.0±1.6 200 + 91.8±1.8 400 + 91.6±3.3 400 + 90.4±1.1

As a result, as shown in FIG. 2, it was confirmed that both the hot water extract and ethanol extract of Chopi tree were not cytotoxic.

2-2: NO generation inhibition ability measurement

In the present invention, in order to confirm the anti-inflammatory effect for each of the hot water extract and ethanol extract obtained in Example 1, the effect of inhibiting nitrite (NO) release was measured, and the measurement of nitrite concentration was performed by a known method ( Kim, JJ, et al. , Journal of Ethnopharmacology , 140:213-221, 2012).

Specifically, BV-2 cells were plated at 1×10 ⁴ cells/well in a 96-well plate, and then, in order to analyze the anti-inflammatory efficacy, hot water extract and ethanol extract of Chopi tree fruit were treated by concentration. After 1 hour, the cells were treated with LPS (200 ng/ml), which is a neuroinflammation inducing factor, and then BV-2 cells were stimulated for 24 hours, and the concentration-dependent inhibitory effect of nitrite (NO) produced at this time was analyzed.

Nitrite (NO) was measured using a NO assay kit (Abcam) using a grease reagent, and the measurement method was carried out according to the instruction manual of the kit. 100 µl of the cell culture solution and grease reagent were mixed, reacted for 10 minutes, and observed at an absorbance of 550 nm, and the concentration was finally confirmed using a standard curve.

Efficacy in inhibiting NO generation by concentration of Chopi tree fruit extract Hot water extract Ethanol extract Addition amount (㎍/㎖) LPS (200ng/ml) NO generation (μM) Addition amount (㎍/㎖) LPS (200ng/ml) NO generation (μM) - - 1.6±0.0 - - 1.8±0.1 - + 23.9±2.8 - + 17.7±0.9 20 + 23.2±2.2 20 + 16.8±0.9 100 + 23.1±2.3 100 + 11.4±0.8 200 + 21.5±1.3 200 + 8.2±0.8 400 + 14.1±1.0 400 + 3.4±0.8

As a result, as shown in FIG. 2, it was confirmed that both extracts inhibit the generation of NO (nitric oxide), and the IC ₅₀ value was found to be 459.33 µg/ml for hot water extract and 189.11 µg/ml for ethanol extract.

Confirmation of Efficacy of Ethanol Extract from Ethanol Extract of Chocolatum for Inhibiting NO Generation

In the present invention, when the ethanol extract of Chopi tree fruit was treated with the ethanol extract on BV-2 microglia stimulated with LPS, the degree of inhibition of NO generation and cytotoxicity was once again confirmed, and the experiment was carried out in the same manner as in Example 2. Performed.

Efficacy of Inhibiting NO Generation and Degree of Cytotoxicity by Ethanol Extract Concentration NO generation Degree of cytotoxicity Addition amount (㎍/㎖) LPS (200ng/ml) NO generation (μM) Addition amount (㎍/㎖) LPS (200ng/ml) Cell viability (%) - - 4.3±1.0 - - 100±1.5 - + 25.1±0.8 - + 96.4±6.4 200 - 2.1±1.5 200 - 99.6±2.9 20 + 23.8±1.0 20 + 92.9±10.7 100 + 18.5±2.0 100 + 89.9±12.8 200 + 13.0±2.0 200 + 97.6±7.3

As a result, as shown in FIG. 3, it was confirmed that the ethanol extract of Chopi in the range of 20 to 200 μg/ml did not affect cytotoxicity, and it was also confirmed that NO generation was effectively suppressed.

Efficacy of Ethanol Extract of Chocolatium Fruits to Inhibit Neuroinflammation

4-1: Confirmation of the ability to inhibit the expression of inflammatory response mediators

In the present invention, it was confirmed that the expression of iNOS and COX-2 proteins, which are mediators of inflammatory reactions, was inhibited when the ethanol extract of Chopi tree fruit was treated on BV-2 microglia stimulated with LPS.

First, cells were cultured in each condition according to the presence or absence of ethanol extract and LPS (200 ng/ml) of Chopi tree fruit. After 6 hours, cells were harvested with 50 mM Tris-HCl (pH 80), 1% Nonidet P-40 (NP-40), 150 mM sodium chloride, 0.5% sodium deoxycholate, 0.1% dodecyl After destruction with a buffer containing sodium sulfate (SDS), protease and phosphatase inhibitor cocktails, the protein was attached to the PVDF membrane after electrophoresis with 10% SDS-PAGE gel.

The membrane was reacted with tris-buffered saline (TBS) containing 5% fat-free oil at room temperature for 2 hours. Again, the membrane was reacted overnight at 4°C with the primary antibody and then reacted for 1 hour at room temperature with an antibody to which horseradish peroxidase (HRP) was attached. The membrane after the reaction was completed was imaged using an ECL detection kit and a luminescent image analyzer (LAS-3000, Fujifilm, Japan).

As a result, as shown in FIG. 4, it was confirmed that the expression of iNOS and COX-2 proteins, which are mediators of the inflammatory response, decreased in a concentration-dependent manner of the ethanol extract of Chopi fruit.

In order to confirm more accurately, the amount of each protein was quantified to measure the relative amount of formation for β-actin, and are shown in Table 4 below.

iNOS and COX-2 protein expression levels Added amount
(㎍/mL) LPS
(200ng/ml) iNOS COX-2 - - One One - + 2.4 ± 1.0 1.5 ± 0.3 200 - 0.3 ± 0.0 0.1 ± 0.0 20 + 1.5 ± 1.0 1.6 ± 0.6 100 + 1.1 ± 1.0 1.5 ± 0.6 200 + 0.3 ± 0.3 0.3± 0.2

4-2: inflammatory reaction Mediator And inflammatory cytokines mRNA Manifestation Inhibitory ability Confirm

In the present invention, when treated with ethanol extract of Chopi tree fruit on BV-2 microglia stimulated with LPS, inflammatory response mediators including NOS and COX-2 and IL-1β, IL-6, and TNF-α are included. It was confirmed that mRNA expression of inflammatory cytokines was suppressed.

First, cells were cultured in each condition according to the presence or absence of ethanol extract and LPS (200 ng/ml) of Chopi tree fruit. After 6 hours, total RNA was isolated using TRIzol reagent. After reverse transcription using 2 μg of RNA, cDNA was prepared using Oligo(dT) 18 primer and AccuPowerTM RT Premix. The gene-specific primers and reaction conditions are shown in Table 5.

PCR primer sequence and reaction conditions Primer sequence (5'-3') order
number Product size (bp) Annealing
Temperature(℃) cycle iNOS F: 5'- GAG GTA CTC AGC GTG CTC CA-3' One 444 56 27 R: 5'-AGG GAG GAA AGG GAG AGA GG-3' 2 COX-2 F: 5'-TGA GTG GTA GCC AGC AAA GC-3' 3 319 56 27 R: 5'-CTG CAG TCC AGG TTC ATG G-3' 4 IL-6 F: 5'-TGA TGC ACT TGC AGA AAA CA-3' 5 71 56 25 R: 5'-ACC AGA GGA AAT TTT CAA TAG GC-3' 6 IL-1β F: 5'-TGT GAA ATG CCA CCT TTT GA-3' 7 94 56 25 R: 5'-GGT CAA AGG TTT GGA AGC AG-3' 8 TNF-α F: 5'-AGG GAG AGT GGT CAG GTT GC-3' 9 392 56 25 R: 5'-CAG CCT GGT CAC CAA ATC AG-3' 10 GAPDH F: 5'-ACC ACA GTC CAT GCC ATC AC-3' 11 472 57 25 R: 5'-CCA CCA CCC TGT TGC TGT AG-3' 12

As shown in Figure 5, mRNA expression of inflammatory mediators including iNOS and COX-2 and inflammatory cytokines including IL-1β, IL-6, and TNF-α is also reduced in a concentration-dependent manner by treatment with ethanol extract of Chopi fruit I confirmed that.

4-3: Confirmation of the ability to inhibit phosphorylation, a major signaling mediator of the inflammatory response

The degree of phosphorylation of IκB-α, which regulates the activity of NF-κB, which regulates the expression of inflammatory mediators and cytokines involved in the inflammatory response, was confirmed.

Western blot was performed in the same manner as in Example 4-1, and an anti-IκBα antibody and an anti-p-IκBα antibody (1:1000; Cell Signaling Technology, USA) were used as primary antibodies.

As a result, as shown in FIG. 6, it was confirmed that phosphorylation of IκB-α, a modulator of NF-κB, was also decreased in a concentration-dependent manner by treatment with ethanol extract of Chopidae.

In other words, it was confirmed that the extract of Chopi tree fruit controls neuroinflammation by inhibiting the mRNA expression of inflammatory factors by inhibiting the activity of NF-κB.

Confirmation of cytotoxicity and anti-inflammatory efficacy according to type of chopidae and extraction site

Chopi tree used in the present invention ( Zanthoxylum piperitum ) To confirm the superiority of fruit extract, Zanthoxylum piperitum ) and the leaves and stem extracts of Zanthoxylum coreanum were confirmed to have cytotoxic and anti-inflammatory effects.

1 ℓ of methanol (99.9%, HPLC grade) was added to 1 kg of dried leaves and stems of Chopi trees and Chopi trees, and then sonicated for 3 days (45 ℃, 15 minutes), then allowed to stand for 2 hours, 10 times/day. ), and freeze-dried through a concentration process.

The degree of anti-inflammatory activity and cytotoxicity of the extract obtained above was performed in the same manner as in Example 2.

As a result, as shown in FIG. 7, it was confirmed that the Chopi tree leaf extract, the Chopi tree leaf and the stem extract suppressed NO generation, but showed toxicity to BV-2 cells. In addition, as shown in FIG. 8, when the chopi tree leaf extract and chopi tree stem extract were treated by concentration, it was confirmed that, in particular, the chopi tree leaf extract showed remarkable cytotoxicity, and as shown in FIG. 9, It was also confirmed that the leaf extract and the stem extract of C. chinensis showed cytotoxicity at high concentration.

Results for FIGS. 8 and 9 are shown in Tables 6 and 7 below.

Efficacy in inhibiting NO generation by concentration according to extract type and site Added amount
(㎍/mL) LPS
(200ng/ml) NO generation (μM) Choppy tree
leaf Choppy tree
stem Tree
leaf Tree
stem - - 2.4±0.3 2.4±0.3 1.6±0.1 1.6±0.1 - + 17.7±0.6 17.7±0.6 17.7±0.7 17.7±0.7 50 + 16.6±0.3 17.2±0.7 13.9±0.6 14.7±0.4 100 + 13.2±2.1 14.4±0.2 8.7±0.5 11.5±0.5 200 + 7.5±0.7 9.3±0.6 4.0±1.3 4.8±0.6

Cytotoxicity by concentration according to extract type and site Added amount
(㎍/mL) LPS
(200ng/ml) Cell viability (%) Choppy tree
leaf Choppy tree
stem Tree
leaf Tree
stem - - 100±6.4 100±6.4 100±2.6 100±2.6 - + 102±2.8 102±2.8 97.3±0.7 97.3±0.7 50 + 88.2±4.1 94.5±4.6 86.4±2.1 93.8±6.6 100 + 75.0±9.4 92.2±9.5 83.9±6.0 88.5±3.1 200 + 62.5±6.2 89.4±6.8 69.5±3.8 84.1±3.0

On the other hand, as shown in Figs. 2 and 3, the chopi tree fruit extract of the present invention has been confirmed to exhibit effective anti-inflammatory activity without cytotoxicity at a high concentration, so that the chopi tree fruit extract of the present invention is Alternatively, it was confirmed that it can be applied as a treatment for degenerative brain diseases without side effects compared to the stem extract.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Composition for Preventing or Treating of Degenerative Brain Disease Comprising Extract of Zanthoxylum piperitum Fruit <130> 1064935 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS_F <400> 1 gaggtactca gcgtgctcca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS_R <400> 2 agggaggaaa gggagagagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2_F <400> 3 tgagtggtag ccagcaaagc 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> COX-2_R <400> 4 ctgcagtcca ggttcatgg 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6_F <400> 5 tgatgcactt gcagaaaaca 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6_R <400> 6 accagaggaa attttcaata ggc 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b_F <400> 7 tgtgaaatgc caccttttga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b_R <400> 8 ggtcaaaggt ttggaagcag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-a_F <400> 9 agggagagtg gtcaggttgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-a_R <400> 10 cagcctggtc accaaatcag 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_F <400> 11 accacagtcc atgccatcac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_R <400> 12 ccaccaccct gttgctgtag 20

Claims (10)

Zanthoxylum piperitum ) A pharmaceutical composition for preventing and treating degenerative brain diseases containing fruit extract as an active ingredient.
The pharmaceutical composition for preventing and treating degenerative brain diseases according to claim 1, wherein the chopi tree fruit extract is extracted using water, a lower alcohol having 1 to 4 carbon atoms, or a mixture thereof as a solvent.
The pharmaceutical composition for preventing and treating degenerative brain diseases according to claim 2, wherein the chopi tree fruit extract is extracted using ethanol as a solvent.
The method of claim 1, wherein the degenerative brain disease is dementia, Alzheimer's disease, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, and Parkinson's disease. (Parkinson disease) a pharmaceutical composition for preventing and treating degenerative brain diseases, characterized in that any one selected from the group consisting of.
The method of claim 1, wherein the chopi tree fruit extract
I) inhibition of production of inflammatory mediators including NO, iNOS and COX-2;
Ii) inhibition of production of inflammatory cytokines including IL-1β, IL-6 and TNF-α; And
Iii) Inhibition of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity through inhibition of IκBα phosphorylation; a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, characterized in that to control nerve inflammation.
A health functional food composition for preventing or improving degenerative brain diseases containing chopi tree fruit extract as an active ingredient.
The health functional food composition for preventing or improving degenerative brain diseases according to claim 6, wherein the chopi tree fruit extract is extracted using water, C ₁ to C ₂ lower alcohol or a mixture thereof as a solvent.
The health functional food composition for preventing or improving degenerative brain diseases according to claim 7, wherein the chopi tree fruit extract is extracted using ethanol as a solvent.
The method of claim 6, wherein the degenerative brain disease is dementia, Alzheimer's disease, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, and Parkinson's disease. (Parkinson disease) health functional food composition for preventing or improving degenerative brain disease, characterized in that any one selected from the group consisting of.
The method of claim 6, wherein the chopi tree fruit extract
I) inhibition of production of inflammatory mediators including NO, iNOS and COX-2;
Ii) inhibition of production of inflammatory cytokines including IL-1β, IL-6 and TNF-α; And
Iii) Inhibition of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity through inhibition of IκBα phosphorylation; health functional food composition for preventing or improving degenerative brain diseases, characterized in that it regulates nerve inflammation .

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